Welcome to Peking University 2009 iGEM Team Notebook Page. We put our experiment notes here for reference. Before you read our notes, several points that you need to know are listed below:
Naming Rule
We name the parts from the registry in many ways. Either by the ID from registry like J23100 or by the location of the physical DNA in the 2009 distribution like 1-7A, where 1 mean kit plate 1, 7A is the location inside of the plate 1.
PCR assessment
We usually use Transgen easyTaq SuperMix to do colony PCR. Earlier, we also use TIANGen Master Mix and Transgen Fast Taq to do colony PCR. The Primer is the Standard prefix and suffix. To save Master Mix, we usually use 5ul of the mix and 0.5 ul for each primer, and 4ul ddH2O.
Enzyme Digestion Assessment
If it is not mentioned specifically, we use Takara EcoRI and PstI for assessment. Later NEB enzymes are also used. We actually digest the DNA in a total volume of 20ul, and later to save enzyme and DNA, we change the total volume to 10ul, but the proportion of the components is not changed.
Ligation
Our ligation uses 3 different ligase, Takara, NEB and later Transgen.
For Takara Ligase, we usually ligate overnight in 16 centigrade or for 4 hours.
For NEB ligase, someone prefer to perform in room temperature for 15 minutes, some in 16 centigrade for 1 hour.
Transgen Ligase is used in the same way as NEB one.
Gel Pictures
We use GeneFinder(a kind of DNA Dye) instead of EB, We mix the sample with Genefinder in a 5:1 proportion and then load the sample to the Gel.
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